Since the first human genome sequences were presented, the research has been focused on the discovery of genetic differences between individuals, such as single-base mutations (“single nucleotide polymorphisms”, SNPs). This is of interest because it becomes more and more evident that single-base variations in the genome are associated with different drug tolerances or predisposition for a wide variety of diseases. In the future, the knowledge of medically relevant nucleotide variations could allow to adapt therapies to the individual genetic supply, and treatment with medicaments which are ineffective or even cause side effects could be prevented (Shi, Expert Rev. Mol. Diagn. 1, 363-365 (2001)). It is obvious that developments which enable a time- and cost-efficient identification of nucleotide variations lead to further progress in pharmacogenetics.
SNPs account for the majority of genetic variations in the human genome and are the cause of more than 90% of the differences between individuals (Kwok, Annu. Rev. Genomics Hum, Genet. 2, 235-258 (2001); Kwok and Chen, Curr. Issues Mol. Biol. 5, 43-60 (2003); Twyman and Primrose, Pharmacogenomics 4, 67-79 (2003)). To detect such genetic variations and other nucleic acid variants, such as mutations, various methods can be employed. For example, the identification of a variant of a target nucleic acid can be effected by hybridizing the nucleic acid sample to be analyzed with a hybridization probe specific for the sequence variant under suitable hybridization conditions (Guo et al., Nat. Biotechnol. 15, 331-335 (1997)).
However, it has been found that such hybridization methods fail to meet, in particular, the clinical requirements in terms of the necessary sensitivity of such assays. Therefore, especially PCR has also found broad use in molecular-biological and diagnostic examination methods for the detection of mutations, single-nucleotide polymorphisms (SNPs) and other allelic sequence variants (Saiki et al., Science 239, 487-490 (1988)), wherein a target nucleic acid to be examined in view of the existence of a variant is amplified by a polymerase chain reaction prior to hybridization. As hybridization probes for such assays, single-strand oligonucleotides are usually used. A modified embodiment of such assays includes those which employ fluorescent hybridization probes (Livak, Genet. Anal. 14, 143-149 (1999)). Generally, it is sought to automate methods for the determination of SNPs and other sequence variations (Gut, Hum. Mutat. 17, 475-492 (2001)).
An alternative of sequence variant specific hybridization which is already known in the prior art is offered by the so-called allele-specific amplification (Newton et al., Nucleic. Acids Res. 17, 2503-2516 (1989); Germer et al., genome res. 10, 258-266 (2000); Gibbs et al., Nucleic. Acids Res. 17, 2437-2448 (1989); Wu et al., PNAS 86, 2757-2769 (1989); Ishikawa et al., Hum. Immunol. 42, 315-318 (1995)). In this detection method, already during the amplification, variant-specific amplification primers are employed which usually have a so-called discriminating terminal nucleotide residue at the 3′-terminal end of the primer, which residue is merely complementary to only one specific variant of the target nucleic acid to be, detected. In this method, nucleotide variations are determined by the presence or absence of DNA product after PCR amplification. The principle of allele-specific amplification is based on the formation of canonical or non-canonical primer-template complexes at the end of allele-specific primer probes. At a correctly paired 3′ primer end, the amplification by a DNA polymerase can occur, while at a mismatched primer end, extension should be inhibited.
For example, U.S. Pat. No. 5,595,890 describes such methods for allele-specific amplification and their application for the detection of clinically relevant point mutations, for example, in the k-ras oncogene. U.S. Pat. No. 5,521,301 also describes methods for allele-specific amplification for the genotyping of the ABO blood group system. In contrast, U.S. Pat. No. 5,639,611 discloses the use of allele-specific amplification in connection with the detection of the point mutation responsible for sickle-cell anemia.
However, allele-specific amplification is problematic in that it is characterized by a low selectivity, which necessitates further complicated and thus time- and cost-intensive optimization steps.
Such methods for detecting sequence variants, polymorphisms and mainly point mutations require allele-specific amplification especially when the sequence variant to be detected is deficient as compared with a predominant variant of the same nucleic acid segment (or of the same gene).
For example, such a situation occurs if disseminated tumor cells are to be detected in body fluids, such as blood, serum or plasma, by means of allele-specific amplification (U.S. Pat. No. 5,496,699). For this purpose, DNA is first isolated from body fluids such as blood, serum or plasma, which DNA is composed of a deficiency of DNA from disseminated tumor cells and an excess of DNA from non-proliferating cells. Thus, the mutations in the k-ras gene significant for tumoral DNA must be detected from a few copies of tumoral DNA in the presence of an excess of wild type DNA.
All the methods for allele-specific amplification described in the prior art have the disadvantage that, despite the use of 3′-discriminating nucleotide residues, a primer extension occurs to a lower extent in the presence of a suitable DNA polymerase even if the target nucleic acid does not exactly correspond to the sequence variant to be detected, i.e., is distinguished therefrom at least by the nucleotide complementary to the nucleotide residue to be discriminated. This leads to false-positive results especially if a particular sequence variant is to be detected in an excess background of nucleic acids containing another sequence variant. As mentioned above, this is the case, for example, in the detection of particular k-ras alleles as indicators of disseminated tumor cells. Another disadvantage of the known methods is the fact that a 3′-terminally discriminating oligonucleotide residue must be used at any rate. The main reason for the disadvantages of these PCR-based methods is the incapability of the polymerases employed in these methods to sufficiently discriminate between base mismatches. Therefore, it has not yet been possible by PCR to directly obtain unambiguous information about the presence or absence of a mutation. To date, further time- and cost-intensive purification and analytical methods have always been necessary for an unambiguous diagnosis of such mutations. Therefore, novel methods which enable an enhancement of the selectivity of allele-specific PCR amplification will have a significant impact on the reliability and robustness of direct SNP analysis by PCR.
On the other hand, a number of modifications have already been described in the protein sequence of DNA polymerases I. Thus, U.S. Pat. No. 6,329,178 mentions DNA polymerase mutants with altered catalytic activity in which there were mutations in the A motif (the highly conserved sequence DYSQIELR (SEQ ID NO:38)). In addition, Minnick, T. et al., J. Biol. Chem. 274, 3067-3075 (1999), describe a wide variety of E. coli DNA polymerase I (Klenow fragment) mutants in which alanine exchanges have been performed. Part of the mutants described exhibit a higher polymerase accuracy as compared to the wild type. One of the mutants mentioned is H881A; particular properties of these mutants with respect to the other mutants described are not stated.
Therefore, it was the object of the present invention to provide sequence variants with enhanced specificity by means of which a sequence variant specific detection method is enabled.